Bacterial Transformation

Nakita McNeil

Independence High School, Manhattan

Summer Research Program for Science Teachers

Summer 2009

Grade Level: 10^th and 11^th grades

Unit: Genetic Engineering, Biotechnology

Objective: Students will be able to used basic scientific inquiry to solve a problem, transform DNA, watch the growth of microscopic species, utilize a natural luminescent to fuel an experiment, learn how plasmids operate, employ good lab practices with microscopic organisms, use the scientific method to create a controlled experiment.

Expected time: student procedure 3 (45minute classes)

Aim: Can we transfer new DNA into existing organisms?

Background (mini lesson)

To transform means to add free DNA into an already constructed strand of DNA, all to provide instructions for that DNA to make a desired protein.

Who is Frederick Griffith?

There are naturally acting transformants (bacteria) that have surface proteins that bind to DNA in the environment and transport it into the cell. Once inside the cell if the new DNA is close in similarity of sequences to the existing DNA, it achieves the ability to substitute specific regions of the bacterium’s DNA. This is known as recombination. Some bacteria that undergo transformations naturally are: Neisseria gonorrhea (the causative agent of gonorrhea, Streptococcus pneumoniae (causes pneumonia), and Haemophilus influenza (causative agent of childhood meningitis). But if we subject bacterium to artificial conditions then we have the ability to force bacteria to take up the new free DNA. When cells are in the state to take up new DNA we coin that stage as competent. Once DNA is taken into the cells, we encourage the possibility of expression of genes.

To avoid the possibility of DNA being lost while we are incorporating it into dissimilar cells we transform host cells with plasmid DNA. A plasmid is a small, circular piece of double stranded DNA. Plasmids naturally occur in bacteria and yeast and are used within the laboratory world to introduce foreign DNA into organisms. Plasmids have origins of replication which allows the host cell to easily replicate upon division without recombination.

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pGREEN plasmid (Contains a mutant strain of GFP, that turns bacteria yellow green in light and fluorescent colonies in UV light.) (p-GLO, Carolina Biological)

Beta-lactamase- selectable marker

Mutant GFP fusion gene – Color marker

Ampicillin resistant, yellow green colonies- Phenotype of transformants

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Have students make predictions of prepared plates (LB-plasmid, LB+plasmid, LB/amp-plasmid, LB/amp+plamid)

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Materials (8 stations)  

E. coli culture

Vial of plasmid DNA at a concentration of 0.005μg/μl (pGREEN)

8 vials of sterile calcium chloride (50mM)

8 vials of sterile LB broth, 3ml each

16 15ml sterile transformation tubes

48 sterile 1ml pipettes, individually sealed or micropipette (10μl-1000μl) and tips

1 bottle of glass beads

28 sterile transfer loops

1 wire inoculating loop

40 sterile Petri plates 

2 bottles of sterile LB agar, 400ml

Vial of ampicillin solution, 4.0ml at 10mg/ml

8 large beakers half full of cracked ice

Collection container for beads

8 culture tube rack

Bunsen burner or a lighter

Water bath 42^oC

Autoclavable disposable bag, or garbage bag

10% bleach solution

Incubator (optional)

Plastic wrap

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Safety

1. Always reflame the inoculating loop before sitting on the workbench
2. Do not eat or drink in the lab, do not taste the broths or cultures
3. Do not over incubate the plates, it encourages unwanted growth
4. Wipe down lab bench with 10% chloride before and after
5. Wash hands
6. Collect bacterial cultures, tubes, pipettes and treat them with bleach for .5hr and place them in the garbage.

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Procedure (Teacher set up)

1. 16 LB plates and 16 LB plates with ampicillin. Prepare not more than 10 days before the lab. Loosen cap of LB broth and heat in a water bath for 20-30minutes. Mix while heating. Do not overheat; the added ampicillin may be inactivated. The bottle should be held in hand without pain. Label plates accordingly LB agar (LB) LB agar with ampicillin (LB/AMP). Use first bottle of LB to pour 16 plates enough to cover the bottom. With the second bottle add ampicillin and swirl to get a good distribution of antibiotic, and then pour 16 plates of LB/amp.
2. Store prepared plates in a plastic sleeve or a Ziploc and keep refrigerated.

Note: We prepare plates a few days before streaking to ensure they are 100% cooled and solidified.

Streaking starter plates

1. label starter plate E. coli
2. Sterilize wire loop with a Bunsen flame, from tip to stalk. Do not set down
3. Touch the loop into your E. coli vial without touching anything else. Flame the vial cap and replace.
4. In a Z like pattern, run your loop over your cooled LB agar plates
5. Place lid on your treated plate
6. Reflame your loop to prevent cross contamination at students work stations
7. repeat for 7 more LB plates
8. Place complete plates agar up in an incubator for 12-20hr at 30^oC or at room temperature 24-40hr.

Student Procedure  

1. In one sterile 15ml tube label +plasmid , other –plasmid
2. Use a sterile transfer pipet to add 250μl of ice cold calcium chloride
3. place both tubes on ice
4. Use a sterile inoculating loop to transfer isolated colonies of E. coli to +plasmid. Your colony should be round transparent and by itself, not joined with another colony. (just pick off, without removing agar)
5. Suspend into +plasmids, while holding the base with loop face down in media rub between the palms of your hands.
6. Suspend the cells by using a transfer pipet (in and out). Observe no visible clumps
7. Return +plasmid to ice, transfer a colony to the –plasmid, return to ice
8. Use a sterile inoculating loop; add one loopful of plasmid DNA to +plasmid tube mix to suspend. Return to ice for 15minutes
9. While tubes are incubating label plates
   1. LB/amp +plasmid
   2. LB/amp –plasmid
   3. +plasmid or –plasmid
10. Heat shock at 42^oC water bath for 90 seconds.
11. Return back to ice for 1minute
12. Use a sterile pipet to add 250μl LB to each tube. Place in a test tube rack for 5-15 minute recovery
13. Add 100μl of cells from the –plasmid transformation tube to each appropriate plate
    1. Slightly open lid and add 4-6 beads or (sterile glass pipet  burned in a shape of an L)
    2. Back and forth shake the beads (spread solution back and forth with pipet)
    3. Let plates rest with beads for 5 minutes
    4. Remove glass beads by slightly opening the lid and tap out into a container.
14. Use another pipet to add 100μl of cell suspension into +plasmid tubes, spread
15. Wrap plates with paraffin and place in a 37^oC incubator for 24-36 hrs. or at room temperature 48-72 hrs.

Analysis Questions

1. Which plate had the most colonies (count), why?
2. Draw out you plates, while viewed in normal light or UV light
3. Which plate would you look at to determine transformation occurred successfully?
4. What is the phenotype (physical characteristic) of the transformed colonies?